Anthocyanin types of seed coats of Vicia faba were found to differ markedly from that of five Phaseolus vulgaris

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1 ISSN: X CODEN: IJPTFI Available Online through Research Article ANTHOCYANINS OF PHASEOLUS VULGARIS AND VICIA FABA SEED COATS Victor I. Deineka*, Yaroslava U. Kulchenko, Ngo Thi Diem Kieu, Yulia N. Kurkina, Ludmila A. Deineka Belgorod State University, , Belgorod, Pobeda Street, 85, Russia. Received on Accepted on Abstract Anthocyanin types of seed coats of Vicia faba were found to differ markedly from that of five Phaseolus vulgaris unknown varieties. Anthocyanin compositions were determined by means of reversed phase HPLC with diode array and mass-spectrometric detectors. For P. vulgaris well known 3-glucosides as well as 3,5-diglucosides of pelargonidin, cyanidin and delphinidin were accomplished in one case by unexpected 3-sambubiosides of the two last anthocyanidins. In the skin of three V. faba varieties 3-rutinosides of delphinidin series of anthocyanidins (delphinidin, petunidin and malvidin synthesised by methyltransferase action being not active in a former genus) were the main components with small amounts of 3-glucosides. Another two types of derivatives were rather rare: 3- rhamnosides and 3-rhamnosylgalactosides. Key words: Seed coats, anthocyanins, Vicia faba, Phaseolus vulgaris, RP HPLC. Introduction The common bean (Phaseolus vulgaris L.) is one the most popular beans worldwide as a food ingredient. The coloration of the bean skin from red or purple to even black is a consequence of anthocyanin biosynthesis [1]. Reviewing the literature data Takeoka et al [1] noticed that anthocyanin series in bean skin are mainly restricted to biosynthesis of 3-glucodides of six common anthocyanidins, delphinidin (Dp), petunidin (Pt), malvidin (Mv), cyanidin (Cy), peonidin (Pn) and pelargonidin (Pg), in the ratios determined by plant variety; though some 3,5- diglucosides were also reported in the cited literature. Yoshida et al [2] in skin of turtle bean (P. vulgaris) have found delphinidin-3-glucoside (Dp3Glu), petunidin-3-glucoside (Pt3Glu), malvidin-3-glucoside (Mv3Glu), petunidin-3,5- diglucoside (Pt3,5diGlu) and malvidin-3,5-diglucoside (Mv3,5diGlu), while the coloration of sultanipya (P. lunatus) skin was due to peonidin-3-rutinoside (Pn3Rut) and peonidin-3-glucoside (Pn3Glu). The lonely Dp3Glu was synthesized in scarlet bean (P. coccineus). IJPT June-2016 Vol. 8 Issue No Page 14088

2 Though broad bean with black colored seed coat (Vicia faba L.) has been used even in Neolithic Age the information about anthocyanins in the plant is rather restricted. Thus anthocyanins were under investigation among other flavonoids by Nozzolillo et al [3]. In the paper only aglicones of violet seeds were analyzed after hydrolysis proving anthocyanins to be the derivatives of malvidin (47 %), petunidin (30 %), delphinidin (19%) and cyanidin (4 %). In the reddish extract of black and red colored seeds the color remained in proanthocyanin fraction and no anthocyanins were found. It is unexpected because in the black skin of soybean 3-glucosides and 3-galactosides of pelargonidin, cyanidin and peonidin were detected [4]. The latter cited paper is more informative than a lot of others publication on the topic due to HPLC chromatogram presentation showing an exhausting character of anthocyanins determination. The aim the current investigation was the determination of anthocyanins of seed coats of same P. vulgaris and V. faba of some popular in Russia central Chernozem region varieties. Plant material 5 varieties of coloured bean seeds of unknown varieties of P. vulgaris were purchased in local market. 3 varieties of V. faba ( Tzarsky urozhay, Bobchinskiye and Belyye ) were grown in Belgorod Botanical garden in the season of Anthocyanins extraction Extraction of anthocyanins was performed directly from the common beans and from the crushed seed coats of V. faba by maceration in 0.1 M HCl solution in water overnight. Such a long time is necessary not only to a complete extraction of anthocyanins but as far as to convert the chalcone forms of anthocyanins into the flavylium one [5]. Namely due to colored flavylium form anthocyanins may be readily determined against the mixture of other colorless phenolic compounds in the real plant extracts. Anthocyanins partial purification The extracts were separated from solid residue by filtration through the paper filter and partially cleaned up by solid phase extraction (SPE) on the syringe cartridges with C18-silica (DIAPAK C18, BioChemMack ST, RF). The cartridge was firstly activated by passing of 5 ml of acetone (2-3 drops per second), conditioned by passing ml of 0.1 M HCl solution in water, and then the extract was passed until colored eluate appearance. The cartridge was washed by 1 ml of 0.1 M HCl solution in water and anthocyanins were desorbed by solution (2-3 ml), contained 30 vol.% of CH 3 CN, 30 vol.% of HCOOH in water and diluted by water in ratio 1:2 (by volume). This dilution permits to escape peak artefacts appearance during HPLC. IJPT June-2016 Vol. 8 Issue No Page 14089

3 Semipreparative isolation of individual anthocyanins The isolation of individual anthocyanins from partially purified extracts was performed by Shimadzu equipment LC- 20 with spectrophotometric detection on chromatographic column mm Supelcosil C18 (5 mcm) in isocratic mode of elution in eluent water acetonitrile formic acid (80:10:10 by volume). Organic solvent was evaporated under reduced pressure and the residue was dried in FreeZone (Labconco) freeze dry system. Anthocyanins acid hydrolysis The sample was mixed with 20% H 2 SO 4 in water and heated in the boiling water bath for min. After cooling anthocyanins were separated from acid by SPE. Reversed phase HPLC conditions For chromatographic experiments Agilent equipment (Infinity 1200) was used with diode-array (DAD), and massspectrometric (MSD, electrospray ionization, positive ion mode) detectors. The data were handled by ChemStation- 32 program. For chromatography only isocratic modes were used with mobile phases, containing 8 6 vol.% of CH 3 CN, 10 vol.% of HCOOH in water at the flow rate of 0.8 ml/min for column mm SymmetryC18, 3.5 mcm, and at the flow rate 0.15 ml/min for column mm Kromasil 100-5C18 (for mass-spectrometric detection). Column thermostat temperature was settled at 40 o C. Chromatograms were stored after registration at 515 nm. Results and discussions Anthocyanin structure determination For the anthocyanin structure determination of the samples under investigation several approaches were used. 1. The comparison of retention times with that of solutes being separated by semi-preparative HPLC from known anthocyanin sources in at least two different mobile phases composition. The latter is necessary to escape the accidental coincidence of the retention times for different solutes because of the possibility of even anthocyanin sequence of elution exchange is the case of mobile (or stationary) phase alteration in reversed-phase chromatography. For 3-glucosides and 3,5-diglucosides of pelargonidin, cyanidin and delphinidin the perfect source of anthocyanins is Punica granatum fruit [6]. The Hibiscus sabdariffa herbal tea may be used to separate 3-sambubiosides of delphinidin and cyanidin [7]. Mahonia aquifolia fruit extract was utilized for 3-rutinosides of the five common anthocyanidins [8] (Dp, Cy, Pt, Pn and Mv) separation. IJPT June-2016 Vol. 8 Issue No Page 14090

4 2. The electronic spectra registered in the detector cell are also useful for the preliminary anthocyanins differentiation because of noticeable alteration of absorption band shape and maxima position for 3-glycosides, 5-glycosides or 3,5- diglycosides of the same anthocyanidin, Table 1. Table 1. Properties of Phaseolus vulgaris and Vicia faba seed coats anthocyanins. t R, min λ max, nm No. Anthocyanin 6%* 8%* M/z 1 Delphinidin-3-sambubioside (303.0) 2 Delphinidin-3-glucoside (303.0) 3 Cyanidin-3,5-diglucoside (287.0) 4 Cyanidin-3-sambubioside (287.0) 5 Cyanidin-3-glucoside (287.0) 6 Petunidin-3-glucoside (317.1) 7 Pelargonidin-3,5-diglucoside (271.1) 8 Pelargonidin-3-glucoside (271.0) 9 Malvidin-3-glucoside (331.1) 10 Delphinidin-3-rutinoside (303.0) 11 Delphinidin-3-rhamnoside (303.0) 12 Delphinidin-3-rhamnosylgalactoside (303.0) 10 Petunidin-3-rutinoside (317.1) 11 Petunidin-3-rhamnoside (317.1) 12 Petunidin-3-rhamnosylgalactoside (317.1) 10 Malvidin-3-rutinoside (331.1) 11 Malvidin-3-rhamnoside n.d (331.1) 12 Malvidin-3-rhamnosylgalactoside (331.1) vol. % CH 3 CN, 10 vol. % HCOOH in water; vol. % CH 3 CN, 10 vol. % HCOOH in water. 3. Mass-spectra also were used to confirm the solutes identification. For fragmentor voltage 200 V and higher massspectra permits to determine the type of anthocyanidin due to intence formation of fragmented ions, while at low voltage the molecular ion is predominant. 4. Acid hydrolysis was also explored. The sample solution in 10 % H 2 SO 4 was heated in boiling water bath min, the products were separated from acid by SPE. Anthocyanins of Phaseolus vulgaris seed skin Sets of anthocyanins of all 5 common bean varieties under investigation were markedly different, Table 1, Fig.1. For the first four varieties only the ratio between mole fractions determined by peak areas of 3-glucosides of IJPT June-2016 Vol. 8 Issue No Page 14091

5 pelargonidin, cyanidin and delphinidin as well as of 3,5-diglucosides of pelargonidin and cyanidin were the main differentiating parameter. The detection of these compounds is in a full agreement with the literature data. But for the fifth one delphinidin-3-glucoside and cyaniding-3-glucoside were accomplished with 3-sambubiosides of the same aglycons. The latter compounds were previously found only in the immature purple pods and black seeds of yardlong beans (Vigna unguiculata ssp. sesquipedalis L.) [9]. Fig.1. Structure of the six common anthocyanidins glycosides. In the case of complicated mixtures of anthocyanins based upon structures with an obvious difference of spectra maximum wavelength a simple normalization approach to calculate the mole fractions of anthocyanins must be changed, the peak areas must be registered at proper wavelengths for each peak, equat.1: S( i) f ( i) ( i) 100, %, (1) S( i) f ( i) i where S(i) area of peak of substance i on the chromatogram, f(i) corrected coefficient for substance i peak on the chromatogram, that is calculated by eq.2: ( i, max) f ( i), %, (2) ( i, ) det where ε(i, λ max ) extinction of the solute i at λ max, ε(i, λ det ) extinction of the solute i at λ of detector used for chromatogram registration; both values must be determined for spectra registered in a cell of diode array detector. Anthocyanins of Vicia faba seed skin The anthocyanin sets of three V. faba varieties skins were nearly the same but were not like that of P. vulgaris seed, Table 1, Fig.2. There were three types of derivatives for anthocyanins of delphinidin set aglicones. Into the latter set of aglicones three structures differing by the degree of ring B OH-groups methylation may be included: IJPT June-2016 Vol. 8 Issue No Page 14092

6 delphinidin, petunidin and malvidin, Table.1, Fig.2. The results are consistent with the literature data [3]. Hence, in the case of V. faba the activity of methyltransferase is rather pronounced (unlike P. vulgaris). Fig.2(a). Separation of anthocyanins of Phaseolus vulgaris seed coats. Column: mm Symmetry C18, 3.5 mcm. Mobile phase: CH 3 CN HCOOH H 2 O (6 : 10 : 84 vol.), 0.8 ml/min. Detector 515 nm. Anthocyanins: 1- Dp3Glu, 2 Cy3,5diGlu; 3 Pg3,5diGlu; 4 Cy3Glu; 5 Pg3Glu; 6 Dp3Sam; 7 Cy3Sam; 8 Pt3Glu. Fig.2(b). Separation of anthocyanins of Vicia faba seed coats. Column: mm Symmetry C18, 3.5 mcm. Mobile phase: CH3CN HCOOH H2O (6 : 10 : 84 vol.), 0.8 ml/min. Detector 515 nm. Anthocyanins: 1 - Dp3Glu, 2 Dp3RhGal; 3 Dp3Rut; 4 Dp3Rh; 5 Pt3Glu; 6 Pt3RhGal; 7 Pt3Rut; 8 Pt3Rh; 9 Mv3Glu; 10 Mv-3RhGal; 11 Mv3Rut; 12 Mv3Rh. The other results of investigation are reported for the first time: IJPT June-2016 Vol. 8 Issue No Page 14093

7 1) The main components were found to be 3-rutinosides (mole fractions by peak areas were Dp3Rut: 8.8 %, Pt3Rut 15.9 %, Mv3Rut 22.4 %); the structure were elucidated by comparison of retention times of the components with that of Mahonia aquifolium (Pursch) Nutt fruits. 2) A rear glycosylation type leading to 3-rhamnosides was also detected being a specific property of Vicia faba (Dp3Rh 9.9 %, Pt3Rh 3.6 % and Mv3Rh 0.9 %). The position of the glycosylation was confirmed by electronic spectra comparison with that of 3-glucosides. While the sugar moiety is proved to be a rhamnosyl one by massspectrometry. 3) The set of derivatives includes also the simple 3-glucosides (Pt3Glu 0.8 % and Mv3Glu 3.2 %). 4) One more type of derivatives X(i) were rather uncommon. According to mass-spectrum as well as to electronic spectrum these anthocyanins were 3-diglycosides, containing rhamnosyl and hexosyl moieties, being isomer of 3- rutinosides, Table 1. The lower retention time of X in comparison with 3-rutinosides may be the consequence of glucose moiety (of 3-rutinosides) substitution by galactose one. The alteration must lead to retention time decrease like in the same substitution for 3-glucoside 3-galactoside pairs [10]. The mole fractions were Dp3RhGal (+Dp3Glu) 2.6 %, Pt3RhGal 3.0 % and Mv3RhaGal 3.6 %. The data are partially in consistence with published data of anthocyanins determination in other legumes plants [11, 12]: 3-rutinosides were found in another species of Vicia genus [11]. But 3-rhamnosylgalactosides of any anthocyanidins were not reported, though some kaempferol derivatives with 2 -rhamnosylgalactoside moiety in position 3 were found in maturing broad bean pods [12]. Chromatography may be used to separate substances and in some cases to propose the structure of substances. Thus, the possibility of 3-rhamnosylgalactoside existence in V. faba seed coats extract may be confirmed by the detection of small amounts of malvidin-3-galactoside, the relative abundance of the solute rises during hydrolysis procedure of the V. faba seed skin extract. Polymeric anthocyanins also are seen on the chromatograms as a small broad tailed peak starting near the dead time, Fig.2. But the mole fraction of colored polymeric compounds in the extracts of the V. faba seed skin under investigation was not so pronounced as in [3]. According to comparison of peak areas the fraction of this type of substances do not exceeds % for the three cultivars. IJPT June-2016 Vol. 8 Issue No Page 14094

8 Table 2. Anthocyanins of common bean seed skin. Conclusion Mole fraction of anthocanins*, % for Phaseolus vulgaris varieties Delphinidin-3-sambubiside Delphinidin-3-glucoside Cyanidin-3,5-diglucoside Cyanidin-3-sambubiside Cyanidin-3-glucoside Petunidin-3-glucoside Pelargonidin-3,5-diglucoside Pelargonidin-3-glucoside Malvidin-3-glucoside Others Degree of hydroxylation Degree of 5-glucosylation Anthocyanins of Phaseolus vulgaris seed coats under investigation were synthesized by action of flavanoid 3 - hydroxylase (F3 H) as well as of flavanoid 3,5 -hydroxylase (F3,5 H) leading to pelargonidin, cyanidin and delphinidin derivatives, while in the case of Vicia faba seed coat F3,5 H is extremely active to convert all anthocyanins into delphinidin set of derivatives, and ratio between the three types (delphinidin, petunidin and malvidin derivatives) of dependent upon methyltransferase activity being suppressed in the case of P. vulgaris varieties under investigation. The glycosylation types of anthocyanins of the two Fabaceae genus seed coats were also different. References 1. Takeoka, G.R., L.T. Dao, G.H. Full, R.Y. Wong, L.A. Harden, R.H. Edwards, J.D.J. Berrios, Characterization of black bean (Phaseolus vulgaris L.) anthocyanins. Journal of Agricultural and Food Chemistry, 45: Yoshida, K., Y. Sato, R. Okuno, K. Kameda, M. Isobe, T. Kondo, Structural Analysis and Measurement of Anthocyanins from Colored Seed Coats of Vigna, Phaseolus, and Glycine Legumes. Bioscience, Biotechnology, and Biochemistry, 60(4): IJPT June-2016 Vol. 8 Issue No Page 14095

9 3. Nozzolillo, C., L. Ricciardi, V. Lattanzio, Flavonoid constituents of seed coat of Vicia faba (Fabaceae) in relation to genetic control of their color. Canadian Journal of Botany, 67: Lee, J.H., N.S. Kang, S.-O. Shin, S.-H. Shin, S.-G. Lim, D.-Y. Suh, I.-Y. Baek, K.-Y. Park, T.J. Ha. Characterization of anthocyanins in the black soybean (Glycine max L.) by HPLC-DAD-ESI/MS analysis. Food Chemistry. 112: Brouillard, R., J. Lang, The hemiacetal cis-chalcone equilibrium of malvin, a natural anthocyanin. Canadian Journal of Chemistry, 68: Du, C.T., F.J. Francis, Anthocyanins of roselle (Hibiscus sadariffa, L). Journal of Food Science, 38: Zhao, X., Z. Yuan, F. Yanming, Y. Yin, L. Feng, Characterization and evaluation of major anthocyanins in pomegranate (Punica granatum L.) peel of different cultivars and their development phases. European Food Research and Technology, 236(1): Sokół-Łętowska, A., A.Z. Kucharska, K. Wińska, A. Szumny, A. Nawirska-Olszańska, P. Mizgier, D. Wyspiańska, Composition and antioxidant activity of red fruit liqueurs. Food Chemistry, 157: Ha, T.J., M.-H. Lee, C.-H. Park, S.-B. Pae, K.-B. Shim, J.-M. Ko, S.-O. Shin, I.-Y. Baek, K.-Y. Park, Identification and Characterization of Anthocyanins in Yard-Long Beans (Vigna unguiculata ssp. sesquipedalis L.) by High-Performance Liquid Chromatography with Diode Array Detection and Electrospray Ionization/Mass Spectrometry (HPLC-DAD-ESI/MS) Analysis. Journal of Agricultural and Food Chemistry, 58: Deineka, V.I., L.A. Deineka, I.I. Saenko, A.N. Chulkov, A Float Mechanism of Retention in Reversed- Phase Chromatography. Russian Journal of Physical Chemistry A, 89(7): Ishikura, N., S. Ito, M. Shirata, Paper Chromatographic survey of anthocyanins in Leguminosae. III. Identification and distribution pattern of anthocyanins in twenty-two legumes. Botanical Magazine, Tokyo, 91: Tomás-Barberán, F.A., M.M. Garcia-Grau, F. Tomás-Lorente, Flavonoid Concentration Changes in Maturing Broad Bean Pods. Journal of Agricultural and Food Chemistry, 39: Corresponding Author: Victor I. Deineka*, russia@prescopus.com IJPT June-2016 Vol. 8 Issue No Page 14096